Abstract The overall goal of this project is to determine the mechanisms of bystander immunosuppression during visceral leishmaniasis (VL). Over 12 million people currently suffer from leishmaniasis, and approximately 2 million new cases occur each year, making it a major global health problem and a WHO classified neglected tropical disease (NTD) (www.who.int). VL is a potentially life-threatening form of Leishmania infection caused by L. donovani and L. infantum. VL is characterized by dissemination of the parasites to the liver, spleen and bone marrow. Many patients with VL are susceptible to secondary bacterial infections due to immunosuppression and develop sepsis, which contributes to 34% to 75% of total VL deaths. However, the specific mechanisms of VL-associated immunosuppression are not clear. L. donovani parasites inhibits the effector functions of infected host macrophages and/or dendritic cells, however, we have recently discovered that L. donovani- infected host macrophages also induce immunosuppression in uninfected bystander macrophages. Using a methodology recently developed in our lab for visualizing and quantifying TLR4 activation, we found that NF-?B activation and nuclear translocation in response to the TLR4 agonist LPS is significantly impaired not only in L. donovani-infected macrophages but also in uninfected macrophages in culture. These findings lead us to hypothesize that parasitized host phagocytes inhibit TLR4 activation in bystander uninfected macrophages and dendritic cells (DCs) which could contribute to increased susceptibility of VL patients to secondary bacterial infections. In this project, we propose to determine how parasitized-macrophages impair TLR4 activation in uninfected bystander macrophages and dendritic cells (DCs) during L. donovani infection. Aim 1 will examine the role of parasitized macrophages and DCs in suppressing TLR4 activation in uninfected bystander phagocytes using fluorescent parasites and a novel imaging flow cytometry based assay recently developed by our group. Aim 2 will determine the mechanism(s) and clinical relevance of L. donovani-mediated bystander immune suppression. The data generated from this study will provide the foundation for future studies focused on understanding the mechanisms of immunosuppression during L. donovani infection and will enable the development of novel strategies to treat VL as well as prevent secondary bacterial infections and sepsis in VL patients.